Trypanosomes are unicellular eukaryotic flagellates which cause serious disease in man and livestock (sleeping sickness, Chagas' disease). They have an intricate life cycle involving transmission between a mammalian host and an insect vector, the tsetse fly. When parasites are transmitted from the tsetse fly to the mammalian host the parasites experience a temperature elevation from roughly 25 degrees C to 37 degrees C and concomitantly differentiation occurs. The proposed research will elucidate the extent of temperature influences on gene expression and differentiation in Trypanosoma brucei. Transcription in T. brucei and other kinetoplastida is characterized by a number of biochemical peculiarities. Among these, the presence of tandemly repeated genes and trans-splicing of polycistronically transcribed pre-mRNA has hampered the characterization of transcription units and the identification of transcription initiation sites of most protein coding genes. The hsp 70 genes were chosen to define the structure of RNA pol II transcription units and the mechanisms for its regulated expression. In the hsp 70 locus of T. brucei, from 5' to 3', diverged cognate hsp 70 gene 1 is separated by about 6 kb from a cluster of five identical hsp 70 genes (genes 2-6). The hsp 70 genes 2-6 are temperature-sensitively and polycistronically transcribed. Recently, by UV-inactivation of transcription and DNA transfection, evidence has been obtained indicating that promoters could be present in front of each hsp 70 gene. In this proposal, Dr. Lee will 1) determine the mechanisms involved in the differential expression of hsp 70 genes in response to differentiation and temperature changes; 2) identify the transcription initiation sites at T. brucei hsp 70 genes and determine the structure of the primary transcripts; 3) study the function of the hsp 70 promoter(s) and determine the transcription efficiency of individual members of tandemly repeated genes; 4) identify potential cis-acting elements and trans-acting factors involved in the transcription of hsp 70 genes; and 5) initiate the study of potential mechanisms in post-transcription control. The significance of this research is that it will unravel the biological importance and molecular mechanisms by which temperature affects gene expression during parasite development. In addition, using established DNA transfection system of T. brucei, the transcription process of protein coding genes can be dissected.